1. Field of the Invention
The present invention relates to anti-apoptotically active aptamers. The corresponding sequences are shown. The invention describes possible therapeutic and diagnostic applications.
2. Related Technology
Nucleic acid molecules (so-called aptamers) which specifically bind with certain target molecules (antigens), as well as methods for the manufacture and isolation of such aptamers, have already been very thoroughly described in the state of the art.
Such aptamers consisting of single-strand ssDNA or ssRNA possess very high affinities and specificities for the corresponding antigens. Up to now, nucleic acid ligands for metallic ions, organic compounds, peptides, proteins, or even complex structures such as viruses and cells have been isolated (Overview article: Gold et al., Annu. Rev. Biochem. 64 (1995), 763-797; Ellington and Conrad, Biotechnol. Annu. Rev. 1 (1995), 185-214; Famulok, Curr. Opin. Struct. Biol. 9 (1999), 324-329).
Aptamers are thus nucleic acids consisting of DNA or RNA which, due to their spatial structure, are able to bind specifically and with a high affinity to a certain target (Osborne, S. E. and Ellington, A. D., (1997). “Nucleic Acid Selection and the Challenge of Combinatorial Chemistry.” Chem Rev 97(2): 349-370). Over the last years, a number of aptamers against medically relevant target proteins have been identified. Furthermore, it is expected that the use of nucleic acids for therapeutic and diagnostic purposes is now first really starting (Jayasena, S. D. (1999). “Aptamers: an emerging class of molecules that rival antibodies in diagnostics.” Clin Chem 45(9): 1628-1650, Cerchia, L., Hamm, J., et al., (2002). “Nucleic acid aptamers in cancer medicine.” FEBS Lett 528(1-3): 12-16). The reasoning behind this new development involves the advantages which the identification and application of DNA or RNA molecules have in comparison with antibodies—which still dominate therapy and diagnosis in many fields to date.
The method for obtaining monoclonal antibodies (Köhler, G. and Milstein, C., (1975). “Continuous cultures of fused cells secreting antibody of predefined specificity.” Nature 256 (5517): 495-497.) meant a large breakthrough for many fields of modern biology and medicine. In medical research, they play their most prominent role in the diagnostic field but, in the meantime, individual antibodies have also been granted approvals as drugs. If a certain protein, e.g. on the cell surface, has been identified as a target for diagnostic or therapeutic purposes and then a) its presence is to be identified for diagnostic purposes, or b) its function is to be blocked for therapeutic purposes, then to date, in both cases, the development of a monoclonal antibody was the most important and/or the fastest method. The development of low-molecular therapeutic agents is still a very long and tedious process. The disadvantages and limitations in the identification and production of antibodies are well known (Jayasena 1999), e.g. the necessity of carrying out the first immunisation with animal trials; the problem of availability and reproducibility of hybridoma cells over several years; or the high workload and enormous costs of antibody production.
An aptamer can be selected under specifically designed experimental conditions, such as e.g. those which are optimal for a diagnostic method (Jayasena 1999). Aptamers can be stored better over longer periods because, unlike proteins, they are not subjected to an irreversible denaturing but can be thermally renatured at any time. During the selection procedure, aptamers are usually synthesised enzymatically. For production on a larger scale however, they can also be synthesised chemically, therefore, in comparison with the production of monoclonal antibodies, their manufacture can be carried out with a much improved reproducibility (Jayasena 1999).
The modifications which lead to the stabilisation of DNA (Agrawal, S. (1996). “Antisense oligonucleotides: towards clinical trials.” Trends Biotechnol 14(10): 376-387) or RNA (Pieken, W. A., Olsen, D. B., et al. (1991). “Kinetic characterization of ribonuclease-resistant 2′-modified hammerhead ribozymes.” Science 253(5017): 314-317; Ruckman, J., Green, L. S., et al. (1998). “2′-Fluoropyrimidine RNA-based aptamers to the 165-amino acid form of vascular endothelial growth factor (VEGF165). Inhibition of receptor binding and VEGF-induced vascular permeability through interactions requiring the exon 7-encoded domain.” J Biol Chem 273(32): 20556-20567), have become established as routine methods in the synthesis of nucleic acids or their nucleotide precursors, so that today, the first aptamers can be tested in clinical trials (Eyetech Study Group (2002). “Preclinical and phase 1A clinical evaluation of an anti-VEGF pegylated aptamer (EYE001) for the treatment of exudative age-related macular degeneration.” Retina 22(2): 143-152).
Apoptosis is a genetically coded “suicide program” which is induced in eukaryotic cells under certain physiological or pathological conditions. The induction of apoptosis must be extremely precisely regulated because a hyperactivity can lead to a degenerative illness. On the other hand, a reduced apoptosis induction can for example contribute to tumour progression.
Different low-molecular inductors of apoptosis have already been described. An important substance class are tumour cytostatics. However, the way in which these cytostatics or other substances induce apoptosis is not known in most cases.
The induction of apoptosis can for example take place via a series of so-called death receptors, i.e. receptors which contain a “Death Domain” (DD) such as CD95, TNF-RI, DR3, DR4 or DR5, which after binding their ligands induce apoptosis signal paths. For example, after binding the CD95 ligand, the CD95 receptor interacts with the adapter protein FADD/MORT1 whereby the “recruitment” and the activation of the protease FLICE/caspase 8 at the DISC “Death Inducing Signalling Complex” are induced. FADD and FLICE each contain “Death Effector Domains” (DED). The induction of apoptosis via these apoptosis signal paths is also possible from outside for example by administering cytotoxic substances, through radiation, viruses, withdrawal of growth factors or mechanical cell injuries. These possibilities of apoptosis induction are however accompanied by certain disadvantages. For example, the administration of toxins such as cytostatics or the radiation treatment of cancer cells can lead to resistance development and even to injury to normal healthy cells where no apoptosis induction whatsoever is desirable.
In general, the induction of apoptosis is proposed for the treatment, for example, of cancer or for preventing angiogenetic processes etc. Although inductors have already been described in this connection, they still demonstrate a number of disadvantages. For example, cytostatics are accompanied by severe side effects.
Pathological conditions where apoptosis has a negative effect and where suitable medical treatment involves the inhibition of apoptosis is also a subject of discussion.
An example of such a condition is arteriosclerosis. In particular, the inventors have already demonstrated earlier that apoptotic cells occur particularly in areas with arteriosclerotic plaque (especially: endothelial cells, smooth muscle cells), and that this occurrence is further enhanced by the disturbed flow conditions, i.e., it is flow-dependent (Freyberg et al., BBRC, 286, 141-149, 2001).
Also, substances which modulate apoptosis can be used to obtain a positive effect on wound healing. Further conditions under discussion which are associated with higher levels of apoptosis are AIDS, cancer and Alzheimer's disease, systemic lupus erythematosus, rheumatoid arthritis and other chronic inflammatory diseases.
There is therefore a large demand for substances which can have a positive or a negative influence on apoptosis. In the sense of the present invention, such substances which inhibit apoptosis are particularly preferred embodiments. It would be particularly favourable to inhibit the flow or force-dependent apoptosis also seen as a causal factor in arteriosclerosis and wound healing problems. A large demand thus remains for pharmaceutical formulations which contain such substances and which can be administered for the treatment of conditions in which the induction or the inhibition of apoptosis is indicated, in particular for the treatment of arteriosclerosis and the improvement of wound healing, as well as the treatment of AIDS, Alzheimer's disease and cancer.
In the older German patent application DE 101 63 130, the inventors of the present invention disclose peptides which inhibit or induce apoptosis.